Project Summary Chemotactic behaviors, movements toward or away from chemicals in an organism?s environment, play important roles in the lives of bacteria. Chemotaxis enables microbes to form communities such as biofilms, to find and colonize host organisms, and to successfully carry out complex, multi-host life cycles. Better understanding of how bacteria detect and respond to their chemical environment will play an important role in the development of new therapeutic strategies for preventing and treating bacterial infections. The long-term goal of this project is to elucidate, in molecular detail, the in vivo signaling mechanisms of the transmembrane receptors that mediate chemotactic behavior in E. coli, a model system for chemotaxis studies. The serine chemoreceptor Tsr forms stable ternary complexes with two cytoplasmic proteins: CheA, a histidine autokinase, and CheW, which couples CheA to chemoreceptor control. These complexes are organized into highly cooperative arrays, typically located at the cell poles, that produce most of the prodigious signal amplification known to occur in the E. coli chemotaxis pathway. The overall objectives of the next project period are to elucidate the mechanisms involved in three signaling processes: signal transmission within chemoreceptor molecules and allosteric control and kinase control in core receptor signaling complexes. Our overall working hypothesis about signal transmission within chemoreceptor molecules proposes that its structural subelements ? external ligand-binding domain, transmembrane helices, HAMP and methylation helix bundles, and cytoplasmic hairpin tip that interacts directly with CheW and CheA ? transmit and process sensory information through shifts in their dynamic behaviors and stabilities. Neighboring elements are coupled in opposition, such that destabilizing inputs to one element produce stabilizing responses in the other. The interplay of these opposing structural forces poises the receptor molecule to detect and respond to small stimulus inputs. The project will test these signaling ideas by mutationally creating structural changes in Tsr, CheA, and CheW and characterizing their signaling consequences with in vivo serine dose-response assays based on Frster Resonance Energy Transfer (FRET). Collaborations with other groups will provide molecular dynamics simulations to assess the structural and dynamics changes in the mutant proteins, single-cell FRET analyses of receptor responses, homo-FRET studies of receptor response kinetics, and cryo-electron microscopy to examine structural features of the mutant receptor arrays.